Project Summary Lung transplantation is considered a last resort approach for patients with end-stage lung disease. However, even with current therapeutic approaches and immunosuppressive drugs, the 5 and 10-year post transplantation survival rates are only 56% and 32% respectively. The primary culprit for chronic lung transplant failure is the formation of small airway fibrosis, bronchiolitis obliterans (BO), resulting in reduced lung function termed bronchiolitis obliterans syndrome (BOS). Currently, it is unclear which lung residential mesenchymal stromal cell (LRMSC) population is responsible for generating BO. Identifying this cell population and underlying key molecular events responsible for collagen deposition and fibrosis could lead to novel therapeutic approaches. A subpopulation of LRMSCs in the normal lung express the transcription factor FoxF1 and are localized around the bronchi and small airways. Interestingly, throughout the alveoli, collagen1a1 expressing LRMSCs are largely devoid of FoxF1 expression. This and other previous data led to the hypothesis that the FoxF1 expressing LRMSC population is unique in its localization and function. With chronic lung transplant rejection being highly associated with small airway fibrosis we hypothesize that these FoxF1 LRMSCs are the primary culprits in forming fibrosis in BOS patients. In aim 1, the unique characteristics and gene expression profile of FoxF1 and non FoxF1 expressing LRMSCs will be elucidated. This will be performed by utilizing the transgenic FoxF1 promoter driven td Tomato reporter (FoxF1-tdTomato) crossed with a Collagen1?1 promoter driven GFP reporter (Col-GFP) mice to isolate two distinct LRMSC populations. In Aim 2, the contribution of FoxF1 expressing LRMSCs in BOS will be investigated. FoxF1-CreERT2 mice crossed with the Rosa-mTmG reporter will be used for allograft and isograft transplantations. Immunohistochemistry will be used to confirm the presence of FoxF1- CreERT2 labeled cells within fibrotic airways. Flow cytometry analysis and cell sorting followed by RNA seq will be performed on FoxF1-CreERT2 labeled cells in allograft vs isograft lungs at day 14 and 40. Additionally, conditional knockout of collagen 1?1 with the FoxF1-CreERT2 mice followed by allograft lung transplantation will gain insight in the overall contribution of FoxF1 expressing LRMSCs to small airway fibrosis and BOS.